Attenuating bacterial virulence by attenuating bacterial folate transport

ABSTRACT

The invention relates to bacterial infections, vaccines directed against those infections and bacterial vaccines. More particularly, the invention relates to vaccines directed against Streptococcus infections in pigs. The invention provides a ΔFolT mutant of a bacterium having reduced capacity to transport folate, wherein said capacity has been reduced by functionally deleting folate transporter (FolT) function. The invention also provides a method to reduce virulence of a bacterium comprising reducing the capacity of said bacterium to transport folate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Application No.PCT/US2017/061170, filed Nov. 10, 2017, which claims the benefit ofEuropean Application No. 16198361.4, filed Nov. 11, 2016, the entirecontents of which are hereby incorporated by reference herein.

SEQUENCE LISTING

This application contains a sequence listing provided as an ASCII textfile submitted via EFS-Web, the entire contents of which is herebyincorporated by reference in its entirety. The ASCII text file,“SequenceListing.txt”, was created on Jan. 10, 2020 and is 21 kb insize.

TECHNICAL FIELD

The invention relates to bacterial strains, drugs directed againstbacterial infections and bacterial vaccines. More particularly, theinvention relates to vaccines directed against Streptococcus infectionsin pigs.

BACKGROUND

Plants, fungi, certain protists, and most bacteria make folate (VitaminB9) de novo, starting from GTP and chorismate, but higher animals lackkey enzymes of the synthetic pathway and so require dietary folate.Folates are crucial to health, and antifolate drugs are widely used incancer chemotherapy and as antimicrobials. For these reasons, folatesynthesis and salvage pathways have been extensively characterized inmodel organisms, and the folate synthesis pathway in both bacteria andplants has been engineered in order to boost the folate content offoods. Tetrahydrofolate is an essential cofactor for many biosyntheticenzymes. It acts as a carrier of one-carbon units in the syntheses ofsuch critical metabolites as methionine, purines, glycine, pantothenate,and thymidylate. For example, the enzyme ketopantoate hydroxymethyltransferase, encoded by the panB gene, requires a tetrahydrofolatecofactor to synthesize precursors of pantothenate. As tetrahydrofolateis synthesized de novo in bacteria, inhibition of its synthesis killscells. Indeed, two very effective antibiotics, sulfonamide andtrimethoprim, kill bacterial cells by blocking tetrahydrofolateproduction. These two antibiotics, which are often used in combinationwith each other, are commonly prescribed for the treatment of urinarytract infections, enteric infections such as shigellosis, andrespiratory tract infections. The success of these drugs is indicativeof the vulnerability of many pathogenic bacteria to inhibitors oftetrahydrofolate synthesis. Bacteria have a multiple step pathway forthe synthesis of the tetrahydrofolate cofactor. In one branch of thepathway, the metabolites chorismate and glutamine are substrates foraminodeoxychorismate synthase, encoded by the B. subtilis genes, pabAand pabB, which produces 4-amino 4-deoxychorismate. Aminodeoxychorismatelyase, encoded by B. subtilis pabC, then converts 4-amino4-deoxychorismate to para-aminobenzoic acid (PABA), an importantprecursor. In another branch, a number of enzymes, including thoseencoded by B. subtilis mtrA, folA, and folK, produce the precursor2-amino-4-hydroxy-6-hydroxy methyl-7,8-dihydroxpteridine diphosphate.This precursor and PABA are substrates for dihydropteroate synthetase,encoded by the B. subtilis sul gene (homologous to the E. coli dhps andfolP genes), which produces dihydropteroate. Sulfonamides, such assulfamethoxazole, are competitive inhibitors of dihydropteroatesynthase. Dihydropteroate is modified by the bifunctional enzyme encodedby B. subtilis folC to produce dihydrofolate. Finally, DHFR(dihydrofolate reductase), encoded by B. subtilis dfrA, modifies thisdihydrofolate to generate the end product tetrahydrofolate. Trimethoprimis a competitive inhibitor of bacterial DHFRs. This selectivity iscritical, as eukaryotic DHFRs are unimpeded by the antibiotic. Folate ismost probably essential for all sequenced bacteria except M.hyopneumoniae. However, not all bacteria synthesize folate de novo butinstead rely on an external supply. To predict the absence of the denovo synthesis pathway, the HPPK (FolK) and DHPS (FolP) proteins areused as signature proteins. Many bacteria lack homologs of both thesegenes and so almost certainly rely on reducing and glutamylating intactfolates taken up from the environment. These are mainly host-associatedbacteria such as Mycoplasma or Treponema or organisms that live infolate-rich environments such as Lactobacilli.

Streptococcus species, of which there are a large variety causinginfections in domestic animals and man, are often grouped according toLancefield's groups. Typing according to Lancefield occurs on the basisof serological determinants or antigens that are among others present inthe capsule of the bacterium and allows for only an approximatedetermination, often bacteria from a different group show crossreactivity with each other, while other Streptococci cannot be assigneda group determinant at all. Within groups, further differentiation isoften possible on the basis of serotyping; these serotypes furthercontribute to the large antigenic variability of Streptococci, a factthat creates an array of difficulties within diagnosis of andvaccination against streptococcal infections. Lancefield group AStreptococcus (GAS, Streptococcus pyogenes), are common with children,causing nasopharyngeal infections and complications thereof. Group Bstreptococci (GBS) constitute a major cause of bacterial sepsis andmeningitis among human neonates and are emerging as significant neonatalpathogens in developing countries. Lancefield group B Streptococcus(GBS) are also found to be associated with cattle, causing mastitis.Lancefield group C infections, such as those with S. equi, S.zooepidemicus, S. dysgalactiae, and others are mainly seen with horse,cattle and pigs. Lancefield group D (S. bovis) infections are found withall mammals and some birds, sometimes resulting in endocarditis orsepticaemia. Lancefield groups E, G, L, P, U and V (S. porcinus, S,canis, S. dysgalactiae) are found with various hosts, causing neonatalinfections, nasopharyngeal infections or mastitis. Within Lancefieldgroups R, S, and T, (and with ungrouped types) S. suis is found, animportant cause of meningitis, septicemia, arthritis and sudden death inyoung pigs. Incidentally, it can also cause meningitis in man. UngroupedStreptoccus species, such as S. mutans, causing caries with humans, S.uberis, causing mastitis in cattle, and S. pneumonia, causing invasivediseases, such as pneumonia, bacteraemia, and meningitis.

Streptococcus suis is a zoonotic pathogen that is ubiquitously presentamong swine populations in the pig industry. Thirty-three capsularserotypes have been described to date [1] of which serotypes 1, 2, 7, 9and 14 are frequently isolated from diseased pigs in Europe [2]. Strainvirulence differs between and within serotypes: within serotype 2,virulent, avirulent as well as weakly virulent isolates have beenisolated that can be discriminated based on the expression of virulencemarkers, muramidase released protein (MRP) and extracellular factor (EF)[3] and suilysin [4,5]. Nasopharyngeal carriage of S. suis in adult pigsis asymptomatic, whereas in young piglets this increases susceptibilityto S. suis invasive disease, leading to meningitis, arthritis andserositis, and high rates of mortality. In Western countries humansoccupationally exposed to pigs or uncooked pork might also becomeinfected by S. suis although the incidence is very low. Invasive S. suisinfection of humans gives similar clinical signs as in pigs; patientsoften suffer from remaining deafness after recovery [6]. In SoutheastAsia, S. suis, in particular of serotype 2, is considered an emergingpathogen for humans, and is recognized as leading cause of bacterialmeningitis [7-10]. In Southeast Asia, clinical signs of human infectionswith S. suis are reported to be more severe compared to other parts ofthe world, with patients developing toxic shock-like syndrome, sepsisand meningitis. Little is known about the pathogenesis of the diseasecaused by S. suis. Various bacterial components, such as extracellularand cell membrane associated proteins, play a role in the pathogenesis.Moreover, it has been shown that the capsule is an important virulencefactor by enabling these microorganisms to resist phagocytosis. Currentstrategies to prevent S. suis infections in pigs include antibiotictreatment of diseased pigs, combined with vaccination strategies withautovaccines [11]. Although auto-vaccination with bacterin vaccinesagainst serotype 2 has shown to be able to reduce clinical outbreaks onfarms, the same is not true for serotype 9, where autovaccination doesnot seem to protect efficiently [12,13]. Besides the fact that bacterinvaccines are less effective against serotype 9 infections, they can onlyprotect against the serotype present in the vaccine. As mentioned beforehowever, several serotypes can cause disease, thus autovaccines are atemporarily solution to a clinical outbreak. For a long-term solutionagainst S. suis infections, vaccines are required that protect broadlyagainst all (pathogenic) serotypes. A lot of research has been done tofind suitable vaccine candidates, however, no cross protective vaccineis available yet.

THE INVENTION

The invention provides a method to produce a bacterium, preferably foruse in a vaccine, preferably for use in a vaccine to generate protectionagainst a bacterial infection, comprising selecting a parent bacterialstrain generally capable of folate transport and folate synthesis andselecting a bacterium from that parent strain for having a modificationsuch as a mutation, deletion or insertion in the DNA region encoding forthe folate substrate binding protein (in Streptococcus suis known as thefolT gene) of said bacterium and selecting said bacterium for thecapacity to grow to similar rates as said parent strain in vitro but togrow to reduced rates compared with said parent strain in vivo. Theinvention also provides a method to produce a bacterium, preferably foruse in a vaccine, preferably a vaccine for use to generate protectionagainst a bacterial infection, comprising selecting a parent bacterialstrain generally capable of folate transport and folate synthesis andtransforming, preferably by recombinant means, a bacterium from thatparent strain by providing it with a modification such as a mutation,deletion or insertion in the DNA region encoding for the folatesubstrate binding protein (in Streptococcus suis known as the folT gene)of said bacterium and selecting said bacterium for the capacity to growto similar rates as said parent strain in vitro but to grow to reducedrates compared with said parent strain in vivo. The invention alsoprovides a bacterium, a bacterial culture obtainable or obtained with amethod of selecting or transforming according to the invention. It ispreferred that said bacterium as provided herein is classifiable as aFirmicutes, preferably a Streptococcus, more preferably a Streptococcussuis. The invention also provides a composition comprising a bacteriumor a culture of a bacterium capable to grow to similar rates as saidparent strain in vitro but growing to reduced rates compared with saidparent strain in vivo. It is also provided to use such a composition forthe production of a vaccine. Preferably, such a vaccine comprises abacterium or a culture of a bacterium capable to grow to similar ratesas said parent strain in vitro but growing to reduced rates comparedwith said parent strain in vivo.

The invention also provides a method to reduce (attenuate) virulence ofa bacterium, said bacterium preferably capable of de novo folatesynthesis, comprising reducing the capacity of said bacterium totransport folate. The inventors provide a bacterium, herein generallycalled ΔFolT mutant, in particular a Streptococcus suis strain is hereinprovided, wherein said capacity has been strongly reduced byfunctionally deleting folate transporter (FolT) function. Thisbacterium, as provided herein, still has the capacity to produce folatefor its own use by applying its de novo folate synthesis pathways.Having these synthesis pathways intact leaves its capacity to in vitrogrowth (in culture) unaffected, surprisingly it was however shown thatits growth and virulence in the host (in vivo) was strongly reduced.Such a bacterial strain that grows well in vitro but in vivo grows lessthan its parent strain and has associated strongly reduced virulence invivo is very useful as a vaccine strain. Such a strain or mutant asprovided by the invention is, on the one hand, essentially unaffected infolate synthesis and thus able to be grown to high titres and therebyrelatively easy and inexpensive to produce, while on the other hand itis, due to its reduced growth and reduced virulence in its host ascompared to its parent strain, relatively innocuous after in vivoapplication, making it extremely useful as a vaccine directed against abacterial infection.

A prototype ΔFolT mutant strain provided with a modification in the DNAregion encoding for the folate substrate binding protein (inStreptococcus suis known as the folT gene) and having similar growth inculture (in vitro) as its parent strain but having reduced growth invivo as opposed to its parent strain, has been deposited as “CBS 140425Streptococcus suis ΔFolT mutant” at the Centraalbureau voorSchimmelcultures for the purpose of patent procedure under theRegulations of the Budapest Treaty at Aug. 19, 2015. Another prototypeΔFolT mutant strain provided with a modification in the DNA regionencoding for the folate substrate binding protein (in Streptococcus suisknown as the folT gene) and having similar growth in culture (in vitro)as its parent strain but having reduced growth in vivo as opposed to itsparent strain, has been deposited as “CBS 143192 Streptococcus suisΔFolT2 mutant” at the Westerdijk Fungal Biodiversity Institute at Aug.25, 2017.

The capacity of de novo folate synthesis of a bacterium can be easilytested by methods known in the art, such as by testing growth of thebacterium in culture media without folate, in comparison with culturemedia provided with folate, or other methods reviewed in BMC Genomics2007, 8:245 (doi:10.1186/1471-2164-8-245, incorporated herein byreference). Most bacteria have at least two independent pathways toacquire tetrahydrofolate: one following the classical folate synthesispathway, and one fast method using the folate transporter to importfolate. In vitro it is now herein provided that there are sufficientnutrients and energy available using the classical synthesis pathway.Not wishing to be bound by theory but offering a possible explanation ofthe effects found by the inventors, in vivo, when there may be lack ofnutrients and thus energy, it may be a lot harder to invest in THFproduction following the classical pathway. The alternative to importfolate is apparently chosen then. Based on ongoing experiments, wepostulate that expression of folT is a burden for the bacterium,probably due to its high hydrophobicity. In vitro, increased expressionof folT decreases growth rate. This is probably the reason whyexpression of folT is so strictly regulated by the presence of itsriboswitch. It should only be expressed when there is absolutenecessity. In conclusion, there seems to be a balance between nutrientavailability and THF requirement versus the burden of proteinexpression. It is now found herein by the inventors that this balancetips in vitro to one side, increased de novo folate synthesis, and invivo to the other side, increased folate transport. Surprisingly,attenuating (reducing) folate transport in the in vivo route, preferablyknocking out folate transport in the in vivo route by functionallydeleting folate transporter function, reduces bacterial virulence in thehost and not in culture. In a preferred embodiment, the inventionprovides a ΔFolT mutant of a bacterium having reduced capacity totransport folate, wherein said capacity has been reduced by functionallydeleting folate transporter (FolT) function. In particular, theinventors herein provide a method to attenuate (reduce) expressionand/or function of the folate substrate binding protein (FolT) of saidbacterium, in particular by providing a mutation, deletion or insertionin the folT gene of said bacterium or in the promotor of said gene. Sucha mutation, deletion or insertion can be detected by PCR and/or sequenceanalysis, as known in the art. In a particular embodiment of theinvention, a method is provided to knockout the folT gene, attenuating abacterium, such as S. suis, considerably, and making it suitable for invivo use as a vaccine strain that still may be cultured easily in vitro.In another embodiment, the invention provides a method wherein saidvirulence is attenuated by providing a mutation, deletion or insertionin the DNA of said bacterium encoding a transmembrane region of folatesubstrate binding protein FolT, preferably leaving immunogenicity ofFolT essentially intact, most preferably leaving the hydrophilic proteindomains of FolT essentially intact. In another embodiment, the inventionprovides a method wherein said virulence is attenuated by providing amutation, deletion or insertion in the FolT encoding DNA region of saidbacterium encoding a region crucial for substrate binding, said regionin S. suis characterized by a peptide domain having a stretch of aminoacids FYRKP. It is preferred to mutate at least the arginine (R) in theFYRKP stretch to abolish folate binding. In a preferred method of theinvention the bacterium is classifiable as a Firmicutes, preferably aStreptococcus, more preferably a Streptococcus suis. It is preferredthat a ΔFolT mutant according to the invention is having the capacity tosynthesize folate; having these synthesis pathways intact leaves itscapacity to in vitro growth (in culture) unaffected, however, stronglyreduces its virulence in a host (in vivo), making it very suitable forvaccine use. It is preferred that said ΔFolT mutant according to theinvention is having attenuated (reduced or hampered) expression of FolT,for example characterised by reduced expression of FolT per se or byexpression of FolT variant protein with reduced molecular weight, suchas can for example be tested by testing FolT specific nucleotideconstructs of said mutant in in vitro transcription/translation studiesas described in the experimental section herein. In one particularpreferred embodiment, the invention provides a ΔFolT mutant according tothe invention deposited as “CBS 140425 Streptococcus suis ΔFolT mutant”at the Centraalbureau voor Schimmelcultures at Aug. 19, 2015. In anotherparticular preferred embodiment, the invention provides a ΔFolT mutantaccording to the invention deposited as “CBS 143192 Streptococcus suisΔFolT2 mutant” at the Westerdijk Fungal Biodiversity Institute at Aug.25, 2017.

In another particular preferred embodiment, the invention provides aΔFolT mutant strain according to the invention. Any of these depositsmay also be used to provide ΔFolT mutant nucleotide constructs ascontrol constructs in expression studies with further bacterial ΔFolTmutants to study expression of FolT variant gene expression or FolTvariant protein expression. Any of these deposits may also be used toprovide a ΔFolT mutant bacterial culture, or a composition comprisingΔFolT mutant bacterial culture according to the invention. The inventionherewith also provides a bacterium with attenuated virulence obtainableor obtained with a method provided herein, and a culture of such abacterium. Also provided is a composition that comprises a ΔFolT mutantbacterium or a ΔFolT mutant culture according to the invention, and useof such a composition for the production of a vaccine. The inventionalso provides a vaccine comprising a ΔFolT mutant bacterium or a ΔFolTmutant culture as provided herein. In a preferred embodiment, providedis a Streptococcus vaccine strain or vaccine, including a ΔFolT mutantcapable of expressing a non-Streptococcus protein. Such avector-Streptococcus ΔFolT mutant vaccine strain allows, when used in avaccine, protection against pathogens other than Streptococcus. Due toits avirulent character, a Streptococcus vaccine strain or ΔFolT mutantas provided herein is well suited to generate specific and long-lastingimmune responses, not only against Streptococcal antigens, but alsoagainst other antigens expressed by the strain. Specifically, antigensderived from another pathogen are now expressed without the detrimentaleffects of the antigen or pathogen, which would otherwise be harmful tothe host. An example of such a vector is a Streptococcus vaccine strainor ΔFolT mutant wherein the antigen is derived from a pathogen, such asActinobacillus pleuropneumonia, Bordetella, Pasteurella, E. coli,Salmonella, Campylobacter, Serpulina and others. Also provided is avaccine including a Streptococcus vaccine strain or ΔFolT mutant and apharmaceutically acceptable carrier or adjuvant. Carriers or adjuvantsare well known in the art; examples are phosphate buffered saline,physiological salt solutions, (double-) oil-in-water emulsions,aluminumhydroxide, Specol, block- or co-polymers, and others. A vaccineaccording to the invention can include a vaccine strain either in akilled or live form. For example, a killed vaccine including a strainhaving (over) expressed a Streptococcal or heterologous antigen orvirulence factor is very well suited for eliciting an immune response.In certain embodiments, provided is a vaccine wherein the strain isliving, due to its avirulent character; a Streptococcus vaccine strainbased on a ΔFolT mutant, as provided herein, is well suited to generatespecific and long-lasting immune responses. Also provided is a methodfor controlling or eradicating a Streptococcal disease in a population,the method comprising vaccinating subjects in the population with aΔFolT mutant vaccine according to the invention. It was provided hereinthat S. suis has an operon that has an important role in pathogenesisand/or virulence of S. suis. The operon encodes two genes involved infolate acquisition and processing of folate into tetrahydrofolate.Folate is a general term for a group of water soluble B-vitamins, wherefolate refers to various tetrahydrofolate derivatives. These derivativescan enter the main folate metabolic cycle, either directly or afterinitial reduction and methylation to tetrahydrofolate. Folate isessential to all living organisms, both prokaryotes and eukaryotes,making folate metabolism a crucial process. The folT-folC operon seemsto form an escape route to acquire folate under folate-restrictedconditions, like for example in vivo where the host sequesters folatefor its own use. Under these conditions, expression of the folT-folCoperon is induced by the riboswitch. When the folate levels drop,tetrahydrofolate will be released from the riboswitch, allowing it tounfold. This allows initiation of translation by the release of theribosomal binding site. Expression of folT-folC allows S. suis to importfolate directly by the folate transporter complex, and subsequentprocess folate into tetrahydrofolate by folC. Since folate is criticalfor nucleotide synthesis, acquisition of folate has a direct effect onthe growth rate of S. suis. Decreased growth rates in vivo leads todecreased virulence. By demonstrating that isogenic knockout mutants offolT such as a mutant deposited as CBS 140425 or as CBS 143192 arestrongly attenuated and useful as a vaccine, this finding was furthersupported. The operon is indeed involved in in vivo pathogenesis. Theinvention herewith now provides such mutants and cultures andcompositions and vaccines comprising such a FolT knockout mutant strainhaving reduced virulence. The invention also provides an immunogeniccomposition comprising a bacterium having such reduced virulence, anduse of such a composition the production of a vaccine,

Furthermore, a vaccine comprising a bacterium ΔFolT mutant, such as amutant deposited as CBS 140425 or as CBS 143192, or a culture or acomposition thereof is provided herein. The invention also provides akit for vaccinating an animal, preferably a pig, against a diseaseassociated with a Streptococcus suis infection comprising: a dispenserfor administering a vaccine to an animal, preferably a pig; and a ΔFolTmutant strain, such as a mutant deposited as CBS 140425 or as CBS143192, according to the invention, or culture or composition thereofand optionally an instruction leaflet. To conclude, the inventionprovides a general method to reduce virulence of a bacterium comprisingreducing the capacity of said bacterium to transport folate, the methodin particular applicable wherein said bacterium is capable of de novofolate synthesis. The method of the invention as herein providedcomprises selecting a bacterium having a functional folate substratebinding protein and then attenuating expression and/or function of thefolate substrate binding protein (FolT) of said bacterium, in particularwherein said virulence is attenuated by providing a mutation, deletionor insertion in the folT gene of said bacterium, for example byproviding a mutation, deletion or insertion in the DNA of said bacteriumencoding the promotor of the folT gene. In certain embodiments of theinvention it is preferred to attenuate said virulence by providing amutation, deletion or insertion in the DNA of said bacterium encoding atransmembrane region of folate substrate binding protein FolT. Inanother particular embodiment, said virulence is attenuated by providinga mutation, deletion or insertion in the FolT encoding DNA region ofsaid bacterium encoding a region crucial for substrate binding. Also, amethod to obtain an immunogenic composition or vaccine is providedherein that is applicable to a bacterium wherein said bacterium is aFirmicutes, preferably a Streptococcus, more preferably a Streptococcussuis, more preferably a ΔFolT mutant deposited as “CBS 140425Streptococcus suis ΔFolT mutant” at the Centraal bureau voorSchimmelcultures at Aug. 19, 2015, most preferably a ΔFolT mutantdeposited as “CBS 143192 Streptococcus suis ΔFolT2 mutant” at theWesterdijk Fungal Biodiversity Institute at Aug. 25, 2017. The inventionalso provides a bacterium with attenuated virulence obtainable orobtained with a method to attenuate virulence by attenuating FolTexpression according to the invention, and provides a culture of a sucha bacterium, and a composition comprising such a bacterium havingreduced virulence or a culture of such a bacterium having reducedvirulence, and an immunogenic composition comprising a bacterium havingreduced virulence or a culture of a bacterium having reduced virulence,and provides use of culture such a bacterium having attenuated FolTexpression or a composition of a bacterial culture having attenuatedFolT expression for the production of a vaccine. The invention alsoprovides a vaccine comprising a bacterium having attenuated FolTexpression or a culture of a bacterium having attenuated FolT expressionor comprising a composition of a culture of a bacterium havingattenuated FolT expression. The invention also provides a kit forvaccinating an animal, against a disease associated with a particularbacterium having FolT expression infection comprising a) a dispenser foradministering a vaccine to an animal, and b) an isogenic knock outstrain (mutant) of said particular bacterium having attenuated FolTexpression, or a culture of an isogenic knock out strain of saidparticular bacterium having attenuated FolT expression, or a compositioncomprising a culture of an isogenic knock out strain of said particularbacterium having attenuated FolT expression, and c) optionally aninstruction leaflet. Such a particular bacterium, according to theinvention provided with attenuated FolT expression and having reducedcapacity to transport folate, wherein said capacity has been reduced byfunctionally deleting folate transporter (FolT) function, in generalhave good growth characteristics in culture media, in particular when aΔFolT mutant according to the invention is used that has the capacity tosynthesize folate; having these synthesis pathways intact leaves itscapacity to in vitro growth (in culture) unaffected, however, stronglyreduces its virulence in a host (in vivo), making it very suitable forvaccine use.

FIGURE LEGENDS

FIG. 1 .

[10] depicts the clone that was identified using a complementationstrategy containing two incomplete ORFs (greyish blue arrows) and aputative operon (purple) containing orf2[10] and folC[10] preceded bythe putative promoter of the operon (for clarification only the sequenceof the −35 region (TGGACA) of the putative promoter is depicted in thediagram). Constructs were made that contained either orf2[10] orfo/C[10](purple) preceded by the putative −35 region of the putative promoterregion of the operon (purple). A construct containing orf2 from strainS735 (green) with the −35 region of the putative S735 promoter (TGGTCA)(green) was made. The same construct was mutagenized to contain the −35region of the putative promoter sequence of strain 10 (TGGACA) (purple)yielding orf2[S735] [t488a].

FIG. 2 . In vitro transcription/translation of FolC and ORF2/FolT. Invitro transcription translation of the control construct pCOM1 (lane 1),and constructs pCOM1-V[10] (lane 2), and pCOM1-folc[10] (lane 3). Themolecular weight marker is indicated in kDa on the right. FolCexpression was detected at the expected weight of 46.8 kDa (closedarrowhead), whereas expression of OR2/FolT was detected at a lowermolecular weight, 14 kDa than expected (20.5 kDa) (open arrowhead).

FIG. 3 . Predicted riboswitch for tetrahydrofolate using Rfam.Three-dimensional structuring of RNA suggested a riboswitch in which twoputative ribosomal binding sites (blue arrows) are inaccessible forribosomes due to folding.

FIG. 4 . Clustal W alignment of different FolT sequences. * indicatesidentical amino acids; : indicates conservation between groups ofstrongly similar properties; . indicates conservation between groups ofweakly similar properties. CB=Clostridium bolteae; CP=Clostridiumphytofermentans; AM=Alkaliphilus metalliredigens; TT=Thermoanaerobactertengcongensis; EFM=Enterococcus faecium; EFS=Enterococcus faecalis;LB=Lactobacillus brevis; SM=Streptococcus mutans; SG=Streptococcusgallolyticus; SUB=Streptococcus uberis; SSU=Streptococcus suis P1/7.

Red indicates the small and hydrophobic amino acids (including aromatic−Tyr); blue indicates acidic amino acids; Magenta indicates basic aminoacids and green indicates hydroxyl, sulphydryl, amine and Gly.

FIG. 5 . Folate metabolism in Streptococcus suis.

Schematic presentation of the putative folate metabolism of S. suis.

FIG. 6 . Expression levels of orf2 and folC in S. suis wild-typeisolates and mutants.

Expression level of orf2 and folC in S. suis wild type isolates strain10 (black bars) and S735 (white bars) grown exponentially in Todd Hewitt(panel A); and in strain S735 complemented with empty control plasmidpCOM1 (black bars), with orf2[10] (white bars) or with orf2[S735](hatched bars) grown exponentially in Todd Hewitt (panel B). Expressionlevel of orf2 in S735 complemented with orf2[10], orf2[S735] andorf2[S735] [t488a] after growing in Todd Hewitt until early exponentialphase (EEP) (white bars), exponential phase (EP) (small hatched bars),late exponential phase (LEP) (large hatched bars) and stationary phase(SP) (black bars) (panel C). Expression levels were determined usingqPCR and expressed as relative expression to housekeeping gene recA. Theexperiments were performed in triplicate; error bars indicate standarderror of the mean. Significance was determined by paired t-tests. *p<0.05; ** p<0.01.

FIG. 7 . Predicted 3-dimensional structure for FolT protein of S. suis.

FIG. 8 . Body temperature of piglets after S. suis infection,experiment 1. Averaged body temperatures of piglets (n=5) eitherinfected with wild type strain 10 (pink) or with strain 10ΔfolT (CBS140425) are depicted. Error bars indicate standard error of the mean.

FIG. 9 . Bacteraemia of piglets after S. suis infection, experiment 1.Averaged bacteraemia of piglets (n=5) either infected with wild typestrain 10 (pink) or with strain 10ΔfolT (CBS 140425) (blue) aredepicted. Error bars indicate standard error of the mean.

FIG. 10 . Survival curves of pigs infected with S. suis, experiment 1.Pigs were infected either wild type strain 10 or with strain 10ΔfolT(CBS 140425). Pigs were euthanized when they reached predeterminedhumane end points for ethical reasons. Statistical analysis was doneusing Log-rank (Mantel-Cox) test.

FIG. 11 . Bacteriological examination of piglets infected with S. suis,experiment 1. Pigs were infected either wild type strain 10 or withstrain 10ΔfolT (CBS 140425) Bacteria were enumerated by serial dilutionand plating. Bacterial counts were calculated as CFU/ml. Differentcolours indicated different individual piglets.

FIG. 12 . Survival curves of piglets infected with S. suis, experiment2. Piglets (n=10) were infected either with wild type strain 10 or withstrain 10ΔfolT (CBS 140425) Pigs were euthanized when they reachedpredetermined humane end points for ethical reasons.

FIG. 13 . Body temperature of piglets after S. suis infection,experiment 2. Averaged body temperatures of piglets (n=10) eitherinfected with wild type strain 10 (blue) or with strain 10ΔfolT (CBS140425) (green) are depicted.

FIG. 14 . Locomotion of piglets after S. suis infection, experiment 2.The percentage of positive observations in piglets either infected withwild type strain 10 (blue) or with strain 10ΔfolT (CBS 140425) areshown. Severity locomotion 1: mild lameness; 2: moderately lameness orreluctance to stand; 3: severe lameness (serving as a human endpoint)

FIG. 15 . Consciousness of piglets after S. suis infection, experiment2. The percentage of positive observations in piglets either infectedwith wild type strain 10 (blue) or with strain 10ΔfolT (CBS 140425).Severity Consciousness: 1: depression; 2: apathy; 3: coma

FIG. 16 . Vaccination of pigs with the ΔFolT 2 strain (CBS 143192) andprotection after challenge with S. suis type 2. Pigs were vaccinated atday 1 and 21 with ΔFolT2 strain (CBS 143192). On day 35, the animalswere challenged intraperitoneally (ip) with approximately 2×10⁹ CFU of avirulent S. suis type 2 isolate. For seven days following challenge, theanimals were observed for signs of disease associated with S. suis.Animals found dead or that had to be euthanized prior to off-test foranimal welfare reasons were necropsied. The figure shows the percentageof animals that died or were euthanized following challenge (mortality).

FIG. 17 . Vaccination of pigs with the ΔFolT strain (CSB140425) andprotection after challenge with S. suis type 2.

Pigs were vaccinated at day 1 and 21 with the ΔFolT strain (CBS 140425).On day 36, the animals were challenged intraperitoneally withapproximately 2×10⁹ CFU of a virulent S. suis type 2 isolate. Followingchallenge, the animals were observed for signs of disease associatedwith S. suis for seven days. Animals found dead or that had to beeuthanized prior to off-test for animal welfare reasons were necropsied.The figure shows the percentage of animals that died or were euthanizedfollowing challenge (mortality).

DETAILED DESCRIPTION Introduction

Previously, we used a complementation strategy to identify novelvirulence factors, which might serve as vaccine candidates. Using thisstrategy, a hypervirulent S. suis isolate (S735-pCOM1-V[10]) wasgenerated that causes severe toxic shock-like syndrome in piglets afterinfection resulting in death within 24 h post-infection[14].S735-pCOM1-V[10] was selected from a library of clones generated in aweakly virulent serotype 2 isolate (S735), after transformation withplasmid DNA isolated from around 30,000 pooled clones carrying randomlycloned genomic DNA fragments from a virulent serotype 2 isolate (strain10). Isolates with increased virulence were selected by infectingpiglets with strain S735 containing the plasmid library of genomicfragments from strain 10. One prevalent clone isolated from the infectedpiglets contained a 3 kb genomic fragment from strain 10 designatedV[10] and was demonstrated to be hypervirulent in subsequent animalexperiments. V[10] contained an incomplete open reading frame (ORF),followed by two genes (orf2 and folC) in an operon structure as well asa second incomplete ORF. Assuming that only the full-length ORFs couldcontribute to the hypervirulence of this isolate, we furthercharacterized the orf2-folC-operon. The first ORF in the operon couldnot be annotated and was designated orf2, the second ORF in the operonshowed homology to the gene encoding polyfolylpolyglutamate synthase(FolC). This operon was present in all S. suis serotypes, including theparent strain S735. Strain S735 with low virulence, contained severalsingle nucleotide polymorphisms (SNP) in orf2-folC and the non-codingregions compared to strain 10. Both genes of the operon that increasedthe virulence may be putative virulence factors and, if so, could beputative vaccine candidates. Here we investigated 1) whether thehypervirulence of the orf2-folC-operon is caused by orf2 or by folC orboth and 2) the effect of a single nucleotide polymorfism in thepromotor region of the orf2-folC-operon on virulence.

Materials and Methods Bacterial Strains and Plasmids

S. suis isolates were grown in Todd-Hewitt broth (Oxoid, London, UnitedKingdom) and plated on Columbia blood base agar plates (Oxoid)containing 6% (vol/vol) horse blood. Escherichia coli was grown in LuriaBroth and plated on Luria Broth containing 1.5% (wt/vol) agar. Ifrequired, erythomycin was added at 1 μg ml⁻¹ for S. suis and at 200 μgml⁻¹ for E. coli. S. suis strain S735 complemented with a plasmidcontaining a 3 kb genomic fragment derived from strain 10(S735-pCOM1-V[10]) and the other S. suis strains used in this study havebeen previously described [14] (FIG. 1 ).

Example 1 Complementation of S. Suis Strain S735

S735 was complemented with plasmid pCOM1 containing one of the two ORFsin the V[10] operon (i.e. orf2[10], or folC[10]) preceded by theputative promoter region of the operon from strain 10 or with plasmidpCOM1 containing orf2 and the cognate upstream promoter from strain S735(orf2[S735]) (FIG. 1 ). To construct these plasmids, primers withrestriction sites were designed to amplify orf2[10] or orf2[S735](comE1-comE2), folC[10] (comE4-comE6) or the promoter region of theoperon (comE1-comE3) (Table 1). The resulting PCR products orf2[10] andorf2[S735] were digested using restriction enzymes SacI and BamHI,cloned into pKUN19 [15], digested with the same restriction enzymes andsubsequently cloned into pCOM1, yielding pCOM1-orf2[10] andpCOM1-orf2[S735], respectively. The PCR amplicon of folC[10] wasdigested using restriction enzymes SmaI and BamHI and cloned into pKUN19cleaved with the same restriction enzymes. The PCR product comprisingthe promoter region of V[10] was cloned in front of folC[10] usingrestriction enzymes SacI and SmaI. Subsequently, the complete fragmentof promoter V[10]-folC[10] was digested from pKUN19 using SacI and BamHIand cloned into pCOM1 digested with the same restriction enzymes,yielding pCOM1-folC[10]. To confirm that the fusion product ofpromoter—folC[10] was transcribed, in vitro transcription/translationwas performed using ³⁵S-methionine. A clear band of the molecular weightof FolC (46.8 kDa) was detected demonstrating that the fusion productcould be expressed and translated (FIG. 2 ). All plasmids wereintroduced into S. suis strain S735 by electroporation. In addition,pCOM1-V[10] was introduced into the avirulent serotype 2 strain T15 byelectroporation to yield T15-pCOM1-V[10].

Example 2 Experimental Infection with Complemented Isolates

Experimental infection of caesarean derived germ-free piglets wasperformed as previously described [14]. Prior to infection, germ-freestatus of piglets was confirmed by plating tonsil swabs on Columbia agarplates containing 6% horse blood. Briefly, 4 or 5 one-week-old germ-freepigs were infected intravenously with 10⁶ colony-forming units (CFU) ofS. suis and then immediately orally administered 40 mg kg⁻¹ body weightof erythomycin (erythomycin-stearate, Abbott B. V., Amstelveen, TheNetherlands) twice a day to keep selective pressure on S. suis isolatesharbouring the pCOM plasmids. Infected pigs were monitored twice dailyfor clinical signs and tonsil swabs collected for bacteriologicalanalysis. Pigs were euthanized when clinical signs of arthritis,meningitis, or sepsis were observed after infection with S. suis. Tissuespecimens of CNS, serosae and joints were collected during necropsy,homogenized and bacterial cell counts were determined by plating serialdilutions on Columbia agar plates containing 6% horse blood and 1 μg oferythomycin. To be able to compare results from different animalexperiments included herein, a uniform scoring of non-specific andspecific symptoms was applied to all animal experiments. Non-specificsymptoms included inappetite and depression that were scored 0 (none),0.5 (mild inappetite/depression) or 1 (severe inappetite/depression).Specific symptoms included lameness, central nervous system (CNS)symptoms (locomotive disorders like cycling, or walking in circles;opistotonus; nystagmus), as well as raised hairs, arched back(kyphosis), and shivering, since these are all symptoms of sepsis orserositis. Based on these observation clinical indices were calculatedby dividing the number of observations where either specific ornon-specific symptoms were observed by the total number of observationsfor this parameter. This represents a percentage of observations whereeither specific or non-specific symptoms were observed. A similarapproach was taken for the ‘Fever Index’. Fever was defined as a bodytemperature>40° C. ‘Mean number of days till death’ was used as asurvival parameter. Although animals were euthanized after reachinghumane end points (HEP), the time between inoculation and reaching HEPsis still indicative of severity of infection. It is calculated byaveraging the survival in days from inoculation until death.

Animal experiments with strain CBS 140425 were performed at the premisesof Central Veterinary Institute of Wageningen UR, Lelystad, TheNetherlands (now named Wageningen Bioveterinary Research (WBVR)) andwere approved by the ethical committee of the Central VeterinaryInstitute of Wageningen UR, Lelystad, The Netherlands, in accordancewith the Dutch law on animal experiments (#809.47126.04/00/01 &#870.47126.04/01/01). Animal experiments with strains CBS 140425 and143192 were also performed in accordance with the US law on animalexperiments.

Statistical analyses were performed on clinical indices of the groups(fever index, specific symptoms and non-specific symptoms) using anon-parametric Kruskal-Wallis test, as there was no homogeneity ofvariance among groups. In subsequent analyses, all groups were comparedpairwise to the control group (S735-pCOM1) on all three parameters,using Mann-Whitney U tests. Differences were considered statisticallysignificant at p<0.05. Calculations were performed using SPSS 19 (IBM,New York, USA).

Example 3 Experimental Infection with Strain 10ΔFolT (CBS 140425),Experiment 1

Ten 4-week-old piglets were housed at CVI animal facility in two groupsof five animals. Piglets had ad lib access to feed and fresh water. Alight provided animals with warmth and play material was availablethroughout the experiment. Prior to the start of the experiment, tonsilswabs of piglets were screened by PCR on colonization of S. suisserotype 2. Only PCR-negative piglets were included in the experiment.After ten days, animals were infected intravenously with either 1.1·10⁶CFU of wild type strain 10 or with 9.2·10⁵ CFU mutant strain 10ΔfolT inthe vena jugularis. Prior to infection basal temperatures of pigletswere monitored daily for a period of three days. EDTA blood wascollected prior to infection to obtain pre-infection plasma samples, aswell as basal levels of white blood cell (WBC) numbers. Infected pigswere monitored three times a day at 8 pm, 3 am and 9 am for clinicalsigns. Non-specific symptoms included lack of appetite and depression,whereas, specific symptoms included lameness, central nervous system(CNS) symptoms (locomotive disorders like cycling, or walking incircles; opistotonus; nystagmus), as well as raised hairs, arched back(kyphosis), and shivering, all of which are symptoms of sepsis orserositis. Tonsil and faecal swabs were collected daily forbacteriological analysis. Blood was collected daily for bacteriologicalanalysis, WBC counting and plasma collection. Pigs were euthanized whenclinical signs of arthritis, meningitis, or sepsis were observed afterinfection with S. suis. At necropsy, internal organs (kidney, liver,spleen, peritoneum and pericardium) were bacteriologically screened forS. suis by plating on Columbia agar plates containing 6% horse blood.Organs that were macroscopically affected by S. suis, like purulentarthritis joints, pericarditis or peritonitis were plated in serialdilution to determine the bacterial load. Tissue specimens of theseorgans were fixated in formalin for histological examination. The animalexperiment was approved by the ethical committee of the CentralVeterinary Institute of Wageningen UR, Lelystad, The Netherlands, inaccordance with the Dutch law on animal experiments (#2014011).

Example 4 Experimental Infection with Strain 10ΔFolT (CBS 140425),Experiment 2

In a second experiment, approximately 3-week old piglets (CommercialCross) were used. The piglets had not been vaccinated against S. suis,had been obtained from a PRRSV negative herd, had never receivedmedicated feed and were tonsil swab negative for S. suis serotype 2 byPCR upon enrolment. Treatment groups (10 piglets each) were housedseparately. Animals were inoculated intravenously with either 3.48E+07CFU of wild type strain 10 or with 1.45E+07 of mutant strain 10ΔfolT.The animals were observed once a day for clinical signs of S. suisassociated disease (e.g. increase in body temperature, lameness, andchanges in behaviour) for 7 days. Any animals displaying clinical signsthat reached humane end-points (e.g. CNS signs, debilitating lameness)were euthanized to minimize suffering. Euthanized animals werenecropsied to identify lesions typically associated with S. suisdisease. Animals surviving to the end of the observation period werelikewise euthanized and necropsied.

Example 5a Vaccination of Pigs with ΔFolT2 Strain (CBS 143192) andProtection after Challenge with S. suis Type 2

The study was conducted in commercial cross pigs; on the day of firstvaccination, the pigs were 21±7 days of age. The animals had not beenvaccinated against S. suis, were tonsil swab negative for S. suis type 2by PCR, PRRSV negative by serology and originated from sows that weretonsil swab negative for S. suis type 2 by PCR. The study groups, thevaccination route and dose, the days of vaccination, and the day androute of challenge are listed in Table 6. The media used are describedin Table 7.

On day 34, blood and tonsil swabs were collected from all animals, andthen the strict control animals were moved to a separate airspace whileall other groups were commingled. On day 35, the animals were challengedintraperitoneally (ip) with approximately 2×10⁹ CFU of a virulent S.suis type 2 isolate.

For seven days following challenge, the animals were observed for signsof disease associated with S. suis. Animals found dead or that had to beeuthanized prior to off-test for animal welfare reasons were necropsied.During necropsy, the animals were assessed for macroscopic signstypically associated with S. suis disease and a CNS (i.e. brain) andjoint swab were collected. At off-test, all remaining animals wereeuthanized, necropsied and samples collected.

The preparation of the vaccines and placebo are listed in Table 7.

The preparation of the challenge material is listed in Table 8.

Vaccination with the S. suis ΔFolT mutant reduced the number of animalsthat died or had to be euthanized for animal welfare reasons during thepost-challenge observation period (see Table 9 and FIG. 16 ). Inaddition, vaccination with the ΔFolT reduced findings of severe lameness(ie. the number of animals being unable to stand or being reluctant tostand), as well as findings of apathy in which the animals showed verylimited to no interest in the environment (see Tables 10 and 11).

During necropsy, signs of inflammation in the brain, indicated by thepresence of fibrin and/or fluid, were less frequently observed in ΔFolTvaccinated animals compared to the negative controls (see Table 12).

The S. suis challenge isolate was less frequently recovered from thebrain and the joint swabs collected at necropsy from animals vaccinatedwith the ΔFolT strain compared to the negative controls (see Tables 13and 14).

Example 5b Vaccination of Pigs with Strain 10ΔFolT (CBS 140425) andProtection after Challenge with S. Suis Type 2

The study was conducted in commercial cross pigs, 21+/−5 days at the dayof the first vaccination. The animals had not been vaccinated against S.suis, were tonsil swab negative for S. suis type 2 by PCR, PRRSVnegative by serology and originated from sows that were tonsil swabnegative for S. suis type 2 by PCR. The study groups, the number ofanimals/group at the time of study initiation, the vaccination dose, thedays of vaccination, the vaccination route, the day of challenge and thechallenge route are listed in Table 15.

On day 35, blood and tonsil swabs were collected from all animals andthe strict control animals were euthanized. On day 36, the animals werechallenged intraperitoneally with approximately 2×10⁹ CFU of a virulentS. suis type 2 isolate.

Following challenge, the animals were observed for signs of diseaseassociated with S. suis for seven days. Animals found dead or that hadto be euthanized prior to off-test for animal welfare reasons werenecropsied. During necropsy, the animals were assessed for macroscopicsigns typically associated with S. suis disease and CNS swabs werecollected. At off-test, all remaining animals were euthanized,necropsied and samples collected.

The preparation of the vaccine and placebo is listed in Table 16. Thepreparation of the challenge material is listed in Table 17.

The S. suis FolT mutant reduced the number of animals showing lamenessfollowing challenge, the number of animals showing abnormal behavior(i.e. depression, coma) following challenge as well as the number ofanimals that died or had to be euthanized for animal welfare reasonsduring the post-challenge observation period (see Table 18, 19 and 20and FIG. 17 ).

At off-test (i.e. at day 7 following challenge or upon removal from thestudy due to death or euthansia) the animals were observed for abnormalfindings in the brain (i.e. fibrin, fluid) as well as in the thoraciccavity (i.e. fibrin, fluid, lung congestion, pneumonia). In addition,samples were collected from the brain for the recovery of S. suis. Theresults are listed in Table 21, 22 and 23.

Example 6 cDNA Synthesis and Quantitative PCR RT-PCR

Two hundred ng of RNA was used to synthesize cDNA in a reactioncontaining 25 ng μl⁻¹ random primers (Promega, Madison, Wis., USA), 10mM dNTPs (Promega), 10 mM DTT (Invitrogen), 40 U RNAsin (Promega) andSuperScriptII Reverse Transcriptase (Invitrogen) according tomanufacturer's instructions.

qPCR

cDNA was diluted 20 times for qPCR analysis. Primers were designed usingPrimerExpress software (Applied Biosystems, Foster City, Calif., USA)(Table 1). Each reaction contained 12.5 pmol forward primer, 12.5 pmolreverse primer and POWR SYBR Green PCR Master Mix (Applied Biosystems)according to manufacturer's instructions. qPCR was performed using anAB17500 (Applied Biosystems). GeNorm software (GeNorm) was used todetermine the most stably expressed reference genes. For S. suis recAwas the least variable in expression of the 6 potential reference genes(phosphogelycerate dehydrogenase (pgd), acetyl-coA acetyltransferase(aca), mutS, glutamate dehydrogenase (gdh) tested. Genorm combinesexpression data into a number representing stability of expression,where 1 represents the most stabile gene. Stability numbers for S. suisranged from 1.667 for gdh to 1.217 for recA. The level of expression ofthese reference genes was measured to control for variation in RNA-yieldand RT-reaction conditions. In each qPCR run a standard curve wasincorporated consisting of a vector containing a cloned PCR product ofthe target gene of that reaction. The standard curve consisted of seven10-fold dilutions of the control vector. In this way both the expressionlevel of the target gene and the expression levels of external referencegenes could be calculated from a standard curve. For each reaction waterwas included in place of cDNA or template as a negative control.Analysis was performed using the AB17500 Software (Applied Biosystems).

Sequence Analysis

Sequence reactions and analysis were performed by Baseclear (Leiden, TheNetherlands).

Example 7 Site-Directed Mutagenesis

Site directed mutagenesis was achieved using the Quick-change IIsite-directed mutagenesis kit (Agilent Technologies, La Jolla, Calif.,USA) according to manufacturer's instructions. PCR primers were designedwith the accompanying software (Agilent Technologies) (Table 1). Usingprimers t448a and t488a_antisense the plasmid pCOM-orf2[S735] wasamplified, introducing the desired mutation that changed the −35 regionof the putative promotor region of the orf2-folC-operon of S735 from5′-TGGTCA-3′ to 5′-TGGACA-3′ (FIG. 1 ). The reaction mixture wasdigested using DpnI to inactivate the original template vector andsubsequently transformed to XL-1-blue competent cells (Invitrogen). Toexclude the possibility of introducing PCR errors into the vectorbackbone, the insert of the plasmid (orf2[S735]) was isolated from thetemplate vector after digestion with restriction enzymes BamHI and SacIand cloned into pCOM1 digested with the same restriction enzymes. Theresulting plasmid was introduced into S. suis isolate S735 byelectroporation and transformants were selected on Columbia agarcontaining 1 μg ml⁻¹ erythomycin, yielding S735-pCOM1-orf2[S735][t488a].Sequencing was used to exclude presence of PCR errors in the finalconstruct.

Example 8 Construction Knock-Out Mutant of S. Suis Folate SubstrateBinding Protein (FolT)

An isogenic folT knock out mutant was constructed in strain 10 bydisrupting folT with a Spectinomycin resistance cassette. pCOM1-V[10][14] was digested with BamHI and ligated into BamHI digested pKUNplasmid, yielding pKUN-V[10]. To remove 3′ part of V[10], pKUN-V[10] wasdigested with SphI after which the vector fragment was purified andligated, yielding pKUN-V[10]*. pKUN-V[10]* was partially digested withXmnI, the linear vector fragment was purified and ligated with the bluntend Spectinomycin resistance cassette, yielding pKUN-V[10]*-Spec^(R).For construction of the mutant V[10]*-Spec^(R) was amplified by PCRusing V735-fw and M13-rev. The PCR product was purified using the PCRPurification Kit (Qiagen). The purified PCR-product was used transformS. suis strain 10 using ComS as competence inducer as described byZaccaria et al. [16] to induce homologous recombination. Transformantswere selected on Columbia agar plates containing 6% (vol/vol) horseblood and 100 μg ml⁻¹ spectinomcyin. Double crossovers were checked byPCR and confirmed using Southern blotting. To exclude the possibility ofintroduction of point mutations, chromosomal DNA of the isogenicknockout mutant was isolated and transformed to strain 10. Again mutantswere selected on Columbia agar plates containing 6% (vol/vol) horseblood and 100 μg ml⁻¹ spectinomcyin, and screened by PCR, yieldingstrain 10ΔfolT. This prototype recombinant ΔFolT mutant strain has beendeposited as “CBS 140425 Streptococcus suis ΔFolT mutant” at theCentraalbureau voor Schimmelcultures for the purpose of patent procedureunder the Regulations of the Budapest Treaty at Aug. 19, 2015.

Example 9 ΔFolT Deletion Mutants not Containing the SpectinomycinResistance Gene

A ΔfolT deletion mutant not containing the Spectinomycin resistance genewas constructed as well. For this the thermosensitive shuttle vectorpSET5s (Takamatsu, D., Osaki, M. and Sekizaki, T. 2001. Plasmids 46:140-148) was used. Plasmid pSET5s contains a temperature sensitiveorigin of replication and can be propagated at 37° C. in E. coli, butreplication of the plasmid is blocked above 37° C. in S. suis (Takamatsuet al). pSET5s contains a cloramphenicol resistance gene (Cm) that canbe used for selection of transformants in E. coli as well as in S. suis.A prototype recombinant ΔFolT mutant strain not containing theSpectinomycin resistance gene has been deposited as “CBS 143192Streptococcus suis ΔFolT2 mutant” at the Westerdijk Fungal BiodiversityInstitute for the purpose of patent procedure under the Regulations ofthe Budapest Treaty at Aug. 25, 2017.

To construct a ΔfolT mutant isolate, a PCR product containing the 5′-and 3′-flanking sequences of the folT gene was generated. This fragmentis cloned into pSET5s and Cm resistant transformants are selected at 37°C. in E. coli. The plasmid was then isolated from E. coli and introducedinto S. suis strain 10. Transformants were selected on Columbia agarplates at 30° C. containing Cm. A transformed colony was used toinoculate 1 ml of Todd Hewitt Broth (THB) containing Cm and the culturewas grown overnight at 30° C. The overnight culture was diluted 100-foldin the same medium and was incubated as above until an optical densityat 600 nm of 0.2-0.3 is reached, at which the culture is transferred to38° C. At this temperature, the plasmid is unable to replicate. Thisstep selects for strains in which the plasmid has integrated into thechromosome via a single recombination event. Serial dilutions of thisculture were plated at Columbia horse blood plates containing Cm. Plateswere incubated overnight at 38° C. A colony containing the recombinantplasmid integrated into the chromosome was picked and inoculated into 1ml of Todd Hewitt Broth (THB) with Cm for incubation overnight at 38° C.The culture was diluted 100-fold with Cm-free THB and grown at 28° C.for five subsequent passages. At this temperature, the plasmid is ableto replicate and is excised from the chromosome via a secondrecombination event over the duplicated target gene sequence. Theexcision of the plasmid can yield the wild type genotype or can resultin a folT deletion mutant. Serial dilutions of the culture were platedonto Columbia horse blood plates (without Cm) and incubated overnight at38° C. Single colonies were then replica plated onto Columbia horseblood plates with and without Cm. Cm sensitive colonies were screened byPCR to identify the ΔfolT mutant isolates not containing theSpectinomycin resistance gene.

Hybridization Studies

Chromosomal DNA was isolated from stationary growing S. suis cultures.Two hundred nanogram of purified DNA was spotted onto Genescreen-Plus(Perkin Elmer, USA). Labelling of probes with ³²P, hybridization andwashing was done as described before [17]. PCR products of folT and folCwere used as a probe, whereas a 16S rRNA probe was used as positivecontrol.

Overexpression of FolT Suffices to Induce Hypervirulence in Strain S735

Introduction of a 3 kb genomic fragment from virulent serotype 2 strain10, V[10], increased the virulence of the weakly virulent serotype 2strain S735 [14], creating a hypervirulent isolate (S735-pCOM1-V[10]).All pigs infected with S735-pCOM1-V[10] died within 1 day post infection(p.i.) and a high percentage of the pigs showed severe clinical signs ofdisease (Table 2), whereas nearly all pigs infected with the controlstrain S735-pCOM1 survived throughout the experiment. Clinical indicesdiffered significantly (p≤0.01) between pigs infected withS735-pCOM1-V[10] and S735-pCOM1 (Table 2). As a control, we also testedthe virulence of S735 transformed with a plasmid containing thehomologous 3 kb fragment from strain S735 (S735-pCOM1-V[S735]). A highpercentage of the pigs infected with S735-pCOM1-V[S735] survivedthroughout the experiment. In contrast pigs infected withS735-pCOM1-V[S735] showed significantly more specific clinical signs(p≤0.01) than pigs infected with S735-pCOM1 (Table 2), althoughdifferences in clinical indices for fever and non-specific symptoms werenot significantly different between the groups (p=0.06). Thus, theincreased copy number of V[S735] in S735, due to introduction of plasmidpCOM1-V[S735] increased specific clinical signs of S. suis.Nevertheless, the specific and non-specific clinical signs due toporcine infection with S735-pCOM1-V[10] (p≤0.01) were significantlyincreased compared to pigs infected with S735-pCOM1-V[S735],demonstrating that the introduction of V[10] in strain S735 increasedthe virulence more than introduction of V[S735]. This result indicatedthat hypervirulence of strain S735 pCOM-1-V[10] might be due to thedifferent nucleotide polymorphisms in V[10] compared to V[S735].

To determine if the both the orf2 and the folC-genes are required forthe observed increase in virulence, both genes of the operon obtainedfrom strain 10 preceded by its cognate promoter sequence were introducedseparately into strain S735 to generate strains S735-pCOM1-orf2[10] andS735-pCOM1-folC[10]. Virulence of these isolates was determined in anexperimental infection in piglets, using S735-pCOM1-V[10] and S735-pCOM1as controls. Table 2 shows that pigs infected with S735-pCOM1-V[10] orwith S735-pCOM1-orf2[10] died within one day p.i. with severe clinicalsigns. Infected pigs developed toxic shock-like syndrome that was notobserved using wild-type strain 10 in experimental infections, implyingfragment V[10] and orf2[10] increased virulence of S735 yielding morevirulent isolates than strain 10 [3]. Both specific and non-specificsymptoms were significantly increased (p<0.01) in pigs infected withS735-pCOM1-V[10] or with S735-pCOM1-orf2[10] compared to S735-pCOM1(Table 2).

Bacteriological examination showed that CNS, serosae and joints werecolonized by high CFU of S. suis. In contrast pigs infected withS735-pCOM1-folC[10] or S735-pCOM1 lived throughout the experiment (11days p.i.) showing mild symptoms of infection, like fever. Nosignificant differences in clinical outcome were observed between pigsinfected with S735-pCOM1-folC[10] and with S735-pCOM1. This clearlydemonstrates that introduction of folC[10] does not increase thevirulence of strain S735, whereas introduction of V[10] and orf2[10]increased the virulence of strain S735. Therefore, we concluded that theobserved increased virulence of S735-pCOM1-V[10] compared to S735-pCOM1was attributed to introduction of orf2[10].

In conclusion, both copy number of V[10] and genetic background of theorf2-folC operon seem to be determinative in the virulence of a givenisolate.

Datamining in Silico Data Demonstrates ORF2 is a Substrate BindingProtein Facilitating Folate Transport

Now that the increased virulence was attributed to introduction ofmultiple copies of orf2[10], the putative function of orf2 was sought.In silico analysis of the 5′ intergenic region preceding the orf2-folCoperon revealed the presence of a predicted secondary structure, whichshowed strong homology to a tetrahydrofolate riboswitch (FIG. 3 ).Tetrahydrofolate riboswitches are a class of homologous RNAs in certainbacteria that bind tetrahydrofolate (THF) [18]. It is almost exclusivelylocated in the probable 5′ untranslated regions of protein-coding genes,and most of these genes are known to encode either folate transportersor enzymes involved in folate metabolism. For these reasons, it wasinferred that the RNAs function as riboswitches. THF riboswitches arefound in a variety of Firmicutes, specifically the orders Clostridialesand Lactobacillales, and more rarely in other lineages of bacteria. TheTHF riboswitch was one of many conserved RNA structures found in aproject based on comparative genomics [19]. The relation with folatemetabolism was confirmed by Eudes et al, demonstrating that in S. suisthe gene upstream of folC encoded a folate transporter, FolT [20]. Thisannotation was also applied to the new genome sequence of S. suis,SC070731, where the homologous gene of ssu0135 was annotated to encodefor FolT (GenBank AGG63648.1). A 3-dimensional structure for the folateenergy-coupling factor transport of Lactobacillus brevis was determined[21], leading to the proposal of a multi protein model of the folatetransporter. In this model ORF2/FolT functions as the transmembranesubstrate-specific binding protein. Together with a more generictransmembrane protein and two nucleotide-binding proteins forming theenergy-coupling module this complex facilitates transmembrane transportof folate. Only the substrate-binding protein (FolT) is specific forfolate, the other components are also used for transport of othersubstrates like thiamine or riboflavin. When the protein sequence ofFolT of S. suis was compared to putative FolT sequences of otherorganisms, it was clear that conserved amino acids were also conservedin S. suis [21] (FIG. 4 ). Interestingly, FIG. 4 also shows that thearginine that was extremely conserved in the human folate transporter aswell as in Escherichia coli tetracycline transporters was also conservedin S. suis (Arg99) folT suggesting this residue to be important fortransporters ranging from men to bacteria [22]. Taken together, thesedata strongly suggest that the conserved protein of unknown function,ORF2, identified using a complementation strategy encodes a substratebinding protein facilitating folate transport.

Folate Transport in Streptococcus suis

Sequence analysis of P1/7 (that is highly homologous to the genome ofstrain 10) indicates that S. suis encodes all enzymes required tosynthesize tetrahydrofolate (THF) via the classical folate biosynthesispathway (FIG. 5 ). Based on data available in the KEGG database(www.kegg.jp) folate metabolism in S. suis makes use of the classicalfolate pathways using folE, folQ, folB, folK, folC, folA and substratesGTP, p-aminobenzoate (PABA) and glutamyl (GLU) as depicted in thescheme. However, with the additional genes present on the V[10] operon,S. suis seems also capable of inducing expression of folT to directlyimport folate and using the simultaneous induced expression of theadditional copy of folC, folate can immediately be processed to theendproduct tetrahydrofolate (THF). In this way the combination of folTand folC forms an additional ‘shortcut’ that allows production of THF.The presence of the THF riboswitch upstream of the folT-folC operonsuggests tight regulation of this two-gene operon that might imply thatthe folT-folC operon is only expressed under specific conditions, e.g.folate poor conditions. Based on the results of the animal experimentdescribed above, it is suggested that the folT-folC operon is at leastexpressed under in vivo conditions.

Presence and Expression of FolT in Streptococcus suis

Presence of the folT gene was demonstrated in all S. suis serotypestested with exception of serotypes 32, and 34. However, serotypes 32 and34 were re-assigned to belong to the genus of Streptococcus orisratti,instead of S. suis [1]. So, in conclusion, all S. suis serotypes aredeemed to have the genes encoding FolT and FolC.

Sequence analysis of the putative promoter of orf2 revealed a differenceat one nucleotide position in the −35 region of the putative promoter instrain 10 (TGGACA) compared to strain S735 (TGGTCA) [14]. The effect ofthis SNP on expression levels of orf2 and folC in strains 10 and S735was determined using qPCR analysis. Significantly higher levels ofexpression of orf2 as well as folC were observed in strain 10 comparedto strain S735 (FIG. 6A). This clearly indicates that the SNP in the −35region of the putative promoter affects the transcription of orf2 andfolC. Thereby, it demonstrates that the identified SNP was indeedlocated in the promoter region co-transcribing orf2 and folC in anoperon. Moreover, introduction of pCOM1-orf2[10] into S735 increasedexpression of orf2 31-fold compared to introduction of pCOM1, whereasintroduction of pCOM1-orf2[S735] increased expression of orf2 only5-fold (FIG. 6B). As expected expression levels of folC were similar forboth recombinant strains (FIG. 6B). To confirm that the identified SNPin the −35 region of the promoter is responsible for the differences intranscription of orf2 in strains S735 and 10 the TGGTCA of orf2[S735]was mutated to TGGACA as found in the promoter of orf2[10] (yieldingstrain S735-pCOM1-orf2[S735][t488a]. Transcript levels of orf2 inS735-pCOM1-orf2[S735][t488a] were shown to be similar to that of strainS735-pCOM1-orf2[10] and four-fold higher than that of strainS735-pCOM1-orf2[S735] in different growth phases (FIG. 6C). Bothpromoters are most active early in the growth phase of S. suis whengrown in Todd Hewitt broth (FIG. 6C). Together, these results clearlydemonstrate that in strain 10, the promoter upstream of orf2-folC-operonis stronger than the promoter upstream of this operon in strain S735,due to an SNP in the −35 region.

To determine whether the SNP linked to increased expression of orf2-folCoperon was associated with particular clonal types or serotypes of S.suis the promoter regions of a large collection of isolates weresequenced (Table 3). All isolates used were recently characterized andtyped by CGH and MLST [23]. Based on the sequence data obtained,isolates could be divided in two main groups (Table 3). The strong −35promoter region was exclusively found in serotype 1 and 2 isolates thatbelonged to CGH cluster A and MLST clonal complex 1 and that expressedthe EF-protein. The SNP associated with lower promoter activity wasfound in serotype 7 and 9 isolates belonging to CGH group B (except fortwo), which are all negative for the expression of EF, as well as inweakly virulent isolates of serotype 2 belonging to CGH group A/ClonalComplex 1 (CC1) that were positive for the expression of the larger formof EF protein (EF*). There were two exceptions; serotype 7 isolate(C126), that belongs to CC1 but does not express the EF-proteincontained the SNP linked to a stronger promoter and serotype 7 isolate(7711) which had a different −35 promoter sequence (TTGTCA) for whichthe promoter strength is undetermined. In conclusion, only CC1 isolatesexpressing EF protein (and 1 serotype 7 isolate) contain the SNP linkedto strong promoter activity. As isolates of this combination ofphenotype and genotype are strongly correlated with virulence [23,24],we can conclude that a strong promoter upstream of orf2-folC-operon isassociated with virulent isolates of S. suis. This observation, togetherwith the increased virulence observed after introduction of additionalcopies of folT[10] suggests that increased expression of folT either dueto increased copy number or due to a stronger promoter leads toincreased virulence in S. suis.

Growth of Streptococcus suis with Additional Copies of FolT or withoutFolT in Culture

No significant differences were observed in growth in culture ofStreptococcus suis with additional copies of folT or without afunctional folT in comparison to the parent strain in vitro.

Protein Expression of FolT

Based on the protein sequence of FolT it was predicted that FolT is avery hydrophobic protein with at least 5 transmembrane domains. Homologymodeling (Expacy server) using 6 known FolT structures among which thepublished 3D structure of FolT from Lactobacillus brevis a 3D structurefor FolT of S. suis was predicted (FIG. 7 ).

FolT is Important for Survival In Vivo: Virulence of a FolT Knock-OutStrain 10ΔfolT

Since overexpression of folT in a weakly virulent S. suis strain led toa strong increase of virulence, we hypothesized that FolT plays animportant role in vivo. To test whether this hypothesis is true, anisogenic knock-out was constructed in virulent S. suis strain 10 byinserting an spectinomycin-resistance cassette in the folT gene. SincefolT and folC are in an operon structure, this knock-out will probablyalso be knocked out for the additional copy of folC. To determinewhether folate transport is essential for virulence in vivo, inexperiment 1, ten pigs were intravenously infected with either wild typestrain 10 or knock out strain 10ΔfolT. All pigs responded to theinoculation with an increase of body temperature (FIG. 8 ). However,pigs infected with the wild type strain 10 showed higher temperaturesfor a longer period of time, compared to pigs infected with the knockoutstrain 10ΔfolT. This is also reflected by a difference in fever index(percentage of observations where pigs displayed fever) between bothgroups (p=0.06). This suggests that strain 10ΔfolT is less pyogenic,compared to the wild type strain. This might be a consequence of thefact that significantly fewer bacteria were isolated from the blood ofpiglets infected with strain 10ΔfolT. Only two pigs infected with strain10ΔfolT showed a short bacteraemic period, compared to 5 pigs infectedwith strain 10; pigs infected with strain 10 also had significantlyhigher numbers of bacteria in their blood for a longer period of time(FIG. 9 ). This suggests, strain 10ΔfolT is either cleared moreefficiently from the blood, or is unable to survive in blood. Whiteblood cell counts revealed that pigs infected with wild types strain 10showed a stronger increase of WBCs for a longer period of time. All pigsinfected with strain 10 displayed increased WBCs, whereas only one ofthe pigs infected with strain 10ΔfolT showed increased WBCs. Thecalculated WBC index differs significantly between the groups (Table 5).Survival rates between the two groups differed significantly: pigsinfected with strain 10 had an average survival of 2,6 days postinfection, whereas pigs infected with strain 10ΔfolT survived 6.2 daysp.i. (FIG. 10 ). Although pigs were euthanized when predetermined humaneend points were reached, survival reflects the severity of infection. Asis shown in FIG. 10 , the survival curves differ significantly betweenthe groups. Gross pathology revealed that ⅘ pigs infected with strain 10showed clinical signs specific for a S. suis infection like arthritis,pleuritis, pericarditis or peritonitis, whereas ⅗ pigs infected withstrain 10ΔfolT showed specific clinical signs. Bacteriologicalexamination of all infected organs revealed that more organs werecolonized by higher bacterial loads for the wild type strain 10 comparedto strain 10ΔfolT (FIG. 11 ).

The second animal experiment (experiment 2) generally confirmed the datagenerated in experiment 1. As in the first experiment, the survivalcurves of wild type strain 10 and the strain 10ΔfolT isolate differedsignificantly. In experiment 2, all animals inoculated with strain10ΔfolT survived until the end of the experiment, whereas 60% of theanimals inoculated with strain 10 had to be euthanized in the course ofthe experiment (FIG. 12 ). Moreover, the frequency and severity ofclinical signs (e.g. temperature, locomotion and consciousness; seeFIGS. 13, 14, 15 ) differed considerably between animals inoculated withwild type strain 10 and strain 10ΔfolT. The frequency of grosspathological lesions in joints and peritoneum obtained at necropsy alsodiffered considerably between the wild type and the 10ΔfolT mutantisolate.

Based on the results of the infection experiments in piglets, it wasconcluded that the isogenic knock out mutant strain 10ΔfolT was stronglyattenuated compared to the wild-type strain. This shows that the folatetransporter is required for bacterial survival under in vivo conditions.Taking the result from both studies together, these experiments clearlyshow that the ΔfolT isolate produced almost no mortality, minimalclinical signs, and a reduced frequency of joint inflammation andperitonitis compared to the parent strain. It can therefore be concludedthat a ΔfolT strain is highly attenuated and safe.

Summary Results. A Vaccine Comprising a Bacterium Provided with aModification Such as a Mutation, Deletion or Insertion in the DNA RegionEncoding for the Folate Substrate Binding Protein (a ΔfolT Isolate) ofSaid Bacterium) of a Bacterium Protects Hosts Against Challenge with aVirulent Isolate of Said Bacterium not having Said Modification

The invention provides a method to produce a bacterium, preferably foruse in a vaccine, preferably for use in a vaccine to generate protectionagainst a bacterial infection, comprising selecting a parent bacterialstrain generally capable of folate transport and folate synthesis andselecting a bacterium from that parent strain for having a modificationsuch as a mutation, deletion or insertion in the DNA region encoding forthe folate substrate binding protein (in Streptococcus suis known as thefolT gene) of said bacterium and selecting said bacterium for thecapacity to grow to similar rates as said parent strain in vitro but togrow to reduced rates compared with said parent strain in vivo. Theinvention also provides a method to produce a bacterium, preferably foruse in a vaccine, preferably a vaccine for use to generate protectionagainst a bacterial infection, comprising selecting a parent bacterialstrain generally capable of folate transport and folate synthesis andtransforming, preferably by recombinant means, a bacterium from thatparent strain by providing it with a modification such as a mutation,deletion or insertion in the DNA region encoding for the folatesubstrate binding protein (in Streptococcus suis known as the folT gene)of said bacterium and selecting said bacterium for the capacity to growto similar rates as said parent strain in vitro but to grow to reducedrates compared with said parent strain in vivo. Such a bacterium, asprovided herein, still has the capacity to produce folate for its ownuse by applying its de novo folate synthesis pathways. Having thesesynthesis pathways intact leaves its capacity to in vitro growth (inculture) unaffected, surprisingly it was however shown herein that itsgrowth and virulence in the host (in vivo) was strongly reduced.

Such a bacterial strain that grows well in vitro but in vivo grows lessthan its parent strain and has associated strongly reduced virulence invivo is very useful as a vaccine strain. Such a strain or mutant asprovided by the invention is, on the one hand, essentially unaffected infolate synthesis and thus able to be grown to high titres and therebyrelatively easy and inexpensive to produce, while on the other hand itis, due to its reduced growth and reduced virulence in its host ascompared to its parent strain, relatively innocuous after in vivoapplication, making it extremely useful as a vaccine directed against abacterial infection.

In a first series of experiments herein, approximately three-week oldpiglets (Commercial Cross) that had not been vaccinated against S. suisand had never received medicated feed were used for the efficacy study.The animals were tonsil swab negative for S. suis serotype 2 by PCR uponenrolment and originated from a PRRSV negative herd. The two treatmentgroups were housed separately at the study site.

Upon arrival at the study site, blood and tonsil swabs were collectedfrom all animals. On study day 0, following an appropriate acclimationperiod, one group of the animals were vaccinated with strain 10ΔFolT.Another group of animals was left unvaccinated. The vaccinated animalswere revaccinated on day 21 into the right side of the neck with samedose of the mutant isolate, respectively. After each vaccination, theanimals were observed for local and systemic reactions. On study day 35,blood and tonsil swabs were collected from all animals before theanimals in both groups were challenged. with the challenge strainATCC700794. The animals were observed for signs of S. suis associateddisease (e.g. increase in body temperature, lameness, abnormalbehaviour, CNS signs) for 7 days following the challenge. Animals founddead or that had to be euthanized prior to off-test for animal welfarereasons were necropsied. During necropsy, the animals were assessed formacroscopic signs typically associated with S. suis disease (e.g.inflammation of CNS, joints, thoracic cavity). In addition, a CNS swabwas collected for recovery of the challenge isolate. On day 42, allremaining animals were euthanized, necropsied and sampled as describedabove. Vaccinated animals showed considerably less signs of S. suisdisease after challenge.

A second series of experiments was conducted in commercial cross pigs;on the day of first vaccination, the pigs were 21±7 days of age. Theanimals had not been vaccinated against S. suis, were tonsil swabnegative for S. suis type 2 PRRSV negative by serology and originatedfrom sows that were tonsil swab negative for S. suis type 2. Uponarrival at the study site, blood and tonsil swabs were collected fromall animals. On study day 0, following an appropriate acclimationperiod, one group of the animals were vaccinated into the left side ofthe neck with strain ΔFolT2. Another group of animals was leftunvaccinated. The vaccinated animals were revaccinated on day 21 intothe right side of the neck with the same dose the mutant isolate,respectively. After each vaccination, the animals were observed forlocal and systemic reactions. On day 34, blood and tonsil swabs werecollected from all animals, and then the strict control animals weremoved to a separate airspace while all other groups were commingled. Onday 35, the animals were challenged intraperitoneally (ip) withapproximately a virulent S. suis type 2 isolate.

For seven days following challenge, the animals were observed for signsof disease associated with S. suis. Animals found dead or that had to beeuthanized prior to off-test for animal welfare reasons were necropsied.During necropsy, the animals were assessed for macroscopic signstypically associated with S. suis disease and a CNS (i.e. brain) andjoint swab were collected. At off-test, all remaining animals wereeuthanized, necropsied and samples collected. Vaccination with theΔfolT2 mutant reduced the number of animals that died or had to beeuthanized for animal welfare reasons during the post-challengeobservation period. During necropsy, signs of inflammation in the brain,indicated by the presence of fibrin and/or fluid, were less frequentlyobserved in ΔfolT2 vaccinated animals compared to the negative controls.The S. suis challenge isolate was less frequently recovered from thebrain and the joint swabs collected at necropsy from animals vaccinatedwith the ΔfolT2 strain compared to the negative controls.

REFERENCES

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TABLES

TABLE 1 Primer sequences. Primer name Sequence 5′-3′ Target comE1cgagctcggaagaa orf2[10]-forward-SacI ttggttattgcgcg tg comE2cgggatcccggggg orf2[10]-reverse-BamHI atgacctgttgctt g comE3tcccccgggggagt P-orf2-folC[10]- cgtgtgtattcgac reverse-SmaI agcgg comE4tcccccgggggaca folC[10]-forward-SmaI agcaacaggtcatc ccc comE6cgggatcccggttg folC[10]-reverse-BamHI aatgcccggcaagc c Orf2-fwctacggctggttct S. suis orf2 tctatcgaa Orf2-rev gcaatcggtgtcatS. suis orf2 gataaagg folC-fw gtttgtccgtccat S. suis cggtttpolyfolylpolyglutamate synthase Folc-rev ctggtcggtcgcat S. suis agatgapolyfolylpolyglutamate synthase RecA-fw ggtttgcaggctcgS. suis recombinase A tatgatg RecA-rev accaaacatgacacS. suis recombinase A cgacttttt t488a gaaaggtatagttt Promoter orf2ttagcaagtgg a ca aaatatatagtgtg tgatacaat t488a_ attgtatcacacacPromoter orf2 antisense tatatattttg t cc acttgctaaaaact atacctttcV735-fw tatgcgcaatgacg pKUN-V[10]*-Spec^(R) tagtagaagg M13-revaacagctatgacca pKUN-V[10]*-Spec^(R) tg

TABLE 2 Virulence of complemented S. suis strains in germfree piglets;all strains contained a plasmid (pCOM1) with or without insert.V[10]/V[S735]: original 3 kb fragment from strain 10 or strain S735 thatwas selected from library; orf2[10]: orf2 from V[10]; folC[10]: orf3from V[10]encoding dihydrofolate synthase. Mean Clinical index No. ofpigs in no. of of the group which S. suis No. days Non- was isolated ofDose Mortality^(a) till Morbidity^(b) Specific^(c) specific^(d) Feverfrom Strain pigs (CFU) (%) death (%) symptoms symptoms index^(e) CNSSerosae^(g) Joints S735-pCOM1- 4 10⁶ 100 1 100 100**  100**  38* 4 4 4V[10] S735-pCOM1- 4 10⁶ 100 1 100 100**   66** 29 4 4 4 orf2[10]S735-pCOM1- 4 10⁶ 0 11 0 4 21  1 0 0 0 folC[10] S735-pCOM1 4 10⁶ 0 11 00 21  5 0 0 0 S735-pCOM1- 5 10⁶ 100 1 100 100**  100**  60* 5 5 5V[10]^(f) S735-pCOM1- 5 10⁶ 20 15 100  43** 38 25 1 1 1 V[S735]^(f)S735-pCOM1^(f) 5 10⁶ 20 16 60 14  11 12 1 0 0 T15-pCOM1- 5 10⁶ 0 14 16 416 13 1 1 1 V[10] ^(a)Percentage of pigs that died due to infection orhad to be killed for animal welfare reasons ^(b)Percentage of pigs withspecific symptoms ^(c)Percentage of observations for the experimentalgroup in which specific symptoms (ataxia, lameness of a least one jointand/or stillness) were observed ^(d)Percentage of observations for theexperimental group in which non-specific symptoms (inappetite and/ordepression) were observed ^(e)Percentage of observations for theexperimental group of a body temperature of >40° C. ^(f)Previousexperiments (Smith et al., 2001) were re-analyzed to allow forstatistical comparison between experiments, this re-analysis requirednew stringent definitions of specific and aspecific symptoms asindicated in materials and methods. *p ≤ 0.05 compared to S735-pCOM1 **p≤ 0.01 compared to S735-pCOM1 ^(g)Serosae are defined as peritoneum,pericardium or pleura

TABLE 3 Sequence analysis of the −35 region of the orf2/folC promoteramong various S. suis isolates and serotypes¹ Pheno- CGH Sero- typeclus- Clonal −35 promoter sequence (5′-3′) type MRP² EF³ ter⁴ complexTGGACA TGGTCA TTGTCA 1 − − B 13 1/1 1 S + A 1 4/4 2 − − B 16/29/147 6/62 + − B 28 1/1 2 + * A 1 7/7 2 − * A 1 1/1 2 + + A 1 9/9 7 − − B 29/1 1/8⁵ 6/8 1/8 9 − − B 16 2/2 9 * − B 16 6/6 9 + − B 16 1/1 ¹ S. suisisolates were described in de Greeff et al.[23] ²* indicates an highermolecular weight form of MRP; s indicates a lower molecular weight formof MRP ³* indicates an higher molecular weight form of EF ⁴All isolateswere genotyped using Comparative Genome Hybridization (CGH) [23] ⁵Thisisolate belongs to clonal complex 1 ⁶Number of isolates analysed/numberof isolates with the respective −35 promoter sequence

TABLE 4 Clinical parameters of pigs infected with S. suis., experiment 1Mean no. of No. days of Mortality until Fever WBC Gross Pathology Strainpigs Dose (%) death Index Index Arthritis Pleuritis PericarditisPeritonitis 10 5 1.1 × 100 2.6   47  50  11/20 2/5 2/5 1/5 10⁶ 10ΔfolT 59.6 × 20 6.2** 23^(#) 19*  2/20 1/5 1/5 0/5 10⁵ *p ≤ 0.05 compared to 10**p ≤ 0.01 compared to 10 ^(#)p ≤ 0.1 compared to 10

TABLE 5 Gross-lesions indicating arthritis and peritonitis: % ofpositive observations in wild type strain 10 and in 10ΔFolT mutantisolate challenged animals; experiment 2 10 10ΔFolT Joints 100 20Peritoneum 80 20

TABLE 6 Study design (for study using CBS 143192) ΔfolT2 CFU VaccinationChallenge Group Treatment per dose (D0, D21) (D35) Off-test 1 Strain10ΔfolT2 5.5 × 10⁷ CFU 0.2 mL id 2 mL ip D42 grown in APS media 2 Strain10ΔfolT2 1.4 × 10⁸ CFU 0.2 mL id grown in THB media 3 Strain 10ΔfolT21.4 × 10⁸ CFU 2.0 mL im grown in THB media 4 Placebo vaccine N/A 2.0 mLim [Negative Control] 5 No treatment N/A N/A N/A [Strict Control]

TABLE 7 Vaccine and placebo preparation (for study using CBS 143192)Group Treatment Description 1 Strain On the vaccination day,ACES-buffered Becton Dickinson APS- 10ΔfolT2 TSB media (APS; w/o serum)was inoculated with ΔfolT2 glycerol grown in APS stock and grown withagitation until 0.6 ± 0.1 OD A600 nm. The media culture was centrifugedat 9,000 × g for 5 minutes at 4° C. The supernatant was decanted andthen the cells were washed twice in an equal volume of sterile 1X PBS,pH 7.2. The washed cells were suspended in PBS to an OD A600 nm equal toapproximately 9 log per mL. Approximately 10 mL of the 9 log washedculture was bottled in sterile vials. Aliquots of the treatment weretested for CFU count prior to vaccination and immediately followingvaccination. The vaccine preparations were held on wet ice untiladministration, no longer than 60 minutes. 2, 3 Strain On thevaccination day, Todd Hewitt broth (THB; w/o serum) 10ΔfolT7 wasinoculated with ΔFolT2 glycerol stock and grown with grown in THBagitation until 0.6 ± 0.1 OD A600 nm. The culture was centrifuged mediaat 9,000 × g for 5 minutes at 4° C. The supernatant was decanted andthen the cells were washed twice in an equal volume of sterile 1X PBS,pH 7.2. The washed cells were suspended in PBS to an OD A600 nm equal toapproximately 9 log per mL. Approximately 10 mL of the 9 log washedculture was bottled in a sterile vial for the group 2 treatment. Analiquot of the 9 log washed culture was further diluted to the targetcell concentration in PBS and bottled in a sterile vial for the group 3treatment. Aliquots of each treatment were tested for CFU count prior tovaccination and immediately following vaccination. The vaccinepreparations were held on wet ice until administration, no longer than60 minutes. 4 Placebo Approximately 40 mL of sterile phosphate bufferedsaline (PBS), vaccine pH 7.2 was bottled in a sterile vial and stored at4° C. until use.

TABLE 8 Challenge preparation (for study using CBS 143192) Strain S.suis type 2 BIAH #08-06 (ATCC 700794 derivative) Preparation A singlecolony was inoculated into 10 mL pre-warmed THB + 5% FBS and grownstatically to 0.5 ± 0.1 OD A600 nm. The culture was scaled up to 900 mLin THB + 5% FBS, and grown with agitation to 0.7 ± 0.1 OD A600 nm.Sterile glycerol was added to the culture (10% v/v). Aliquots wereretained for pre-freeze and post-thaw CFU and for purity. The challengewas dispensed into vaccine bottles and stored at −70° C. until use.Prior to use, the culture was thawed in a 37° C. waterbath, then dilutedwith sterile THB + 5% FBS to meet the target concentration of 1 × 10⁹cfu/mL. Aliquots of the treatment were tested for CFU count prior tochallenge and immediately following challenge.

TABLE 9 Percentage of animals that died or were euthanized followingchallenge (mortality) (for study using CBS 143192) # Pigs Groupchallenged Vaccine Mortality 1 14 Strain 10ΔfolT2 - 35.7% 7 log - APS -id 2 11 Strain 10ΔfolT2 - 45.5% 8 log - THB - id 3 11 Strain 10ΔfolT2 -27.3% 8 log - THB - im 4 15 Placebo vaccine - 93.3% Negative Control

TABLE 10 Percentage of animals showing severe lameness followingchallenge (for study using CBS 143192) Percentage of pigs showing # Pigssevere lameness during Group challenged Vaccine observation period 1 14Strain 10ΔfolT2 - 4.2% 7 log - APS - id 2 11 Strain 10ΔfolT2 -  0% 8log - THB - id 3 11 Strain 10ΔfolT2 - 3.3% 8 log - THB - im 4 15 Placebovaccine - 41.7%  Negative Control

TABLE 11 Percentage of animals showing apathy following challenge (forstudy using CBS 143192) Percentage of pigs showing # Pigs apathy duringGroup challenged Vaccine observation period 1 14 Strain 10ΔfolT2 - 21.1%7 log - APS - id 2 11 Strain 10ΔfolT2 - 4.3% 8 log - THB - id 3 11Strain 10ΔfolT2 - 11.7% 8 log - THB - im 4 15 Placebo vaccine - 50.0%Negative Control

TABLE 12 Percentage of animals showing signs of inflammation in brainduring necropsy (for study using CBS 143192) # Pigs Percentage of pigsshowing Group challenged Vaccine inflammation in brain 1 14 Strain10ΔfolT2 - 21% 7 log - APS - id 2 11 Strain 10ΔfolT2 - 45% 8 log - THB -id 3 11 Strain 10ΔfolT2 - 27% 8 log - THB - im 4 15 Placebo vaccine -87% Negative Control

TABLE 13 Percentage of animals from which S. suis was recovered frombrain swabs collected at necropsy (for study using CBS 143192)Percentage of pigs from # Pigs which S. suis was Group challengedVaccine recovered from brain 1 14 Strain 10ΔfolT2 - 35.7% 7 log - APS -id 2 11 Strain 10ΔfolT2 - 27.3% 8 log - THB - id 3 11 Strain 10ΔfolT2 -27.3% 8 log - THB - im 4 15 Placebo vaccine - 93.3% Negative Control

TABLE 14 Percentage of animals from which S. suis was recovered from thejoints swabs collected at necropsy (for study using CBS 143192)Percentage of pigs from # Pigs which S. suis was Group challengedVaccine recovered from the joints 1 14 Strain 10ΔfolT2 - 35.7% 7 log -APS - id 2 11 Strain 10ΔfolT2 - 36.4% 8 log - THB - id 3 11 Strain10ΔfolT2 - 18.2% 8 log - THB - im 4 15 Placebo vaccine - 73.3% NegativeControl

TABLE 15 Vaccination challenge study outline (for study using CBS140425) Inclusion # Level/2 ml Days of Vacc. Challenge Group PigsTreatment dose Treatment Route Day Chall. Route 1 15 Strain 10ΔfolT 1.0× 10¹⁰ 0, 21 i.m. 36 i.p. CFU (first vac) 9.8 × 10⁹ CFU (second vac) 215 Strain 10ΔfolT 9.5 × 10⁹ 0 i.m. 36 i.p. CFU 3 15 Placebo N/A 0, 21i.m. 36 i.p. [Negative Control] 4 5 Strict control N/A N/A N/A N/A N/A

TABLE 16 Vaccine preparation (for study using CBS 140425) GroupTreatment Description 1-2 Strain 10ΔfolT A strain 10ΔfolT glycerol stockwas transferred into Todd- Hewitt Broth (THB) + 5% Fetal Bovine Serum(FBS) and grown statically to 0.5 ± 0.1 OD A600 nm. The culture wasscaled up to 1800 mL in THB + 5% FBS, and grown with agitation to 0.7 ±0.1 OD A600 nm. The culture was concentrated 6X by centrifugation andremoval of supernatant to achieve a 10-log dose. Sterile glycerol wasadded to the concentrated culture (10% v/v). Aliquots were retained forpre-freeze and post- thaw CFU, identity, and purity. The vaccine wasdispensed into vaccine bottles and stored at −70° C. until use. Thevaccine was thawed in a 37° C. water bath and diluted to the intendedtarget concentration using storage media, then held on wet ice untiladministration. 6 Placebo Sterile THB + 5% FBS media, stored at 4° C.until use.

TABLE 17 Challenge preparation (for study using CBS 140425) ChallengeStrain S. suis type 2 BIAH #08-06 (ATCC 700794 derivative) ChallengePreparation A single colony was inoculated into 20 mL pre-warmed THB +5% FBS and grown statically to 0.5 ± 0.1 OD A600 nm. The culture wasscaled up to 900 mL in THB + 5% FBS, and grown with agitation to 0.7 ±0.1 OD A600 nm. Sterile glycerol was added to the culture (10% v/v).Aliquots were retained for pre-freeze and post-thaw CFU and for purity.The challenge was dispensed into vaccine bottles and stored at −70° C.until use.

TABLE 18 Percentage of animals showing lameness following challenge (CBS140425) Percentage of pigs Group # Pigs Vaccine showing lameness 1 13Strain 10ΔfolT - 7.7% 10 logs - 2 dose 2 15 Strain 10ΔfolT - 40.0% 10logs - 1 dose 3 15 Placebo 93.3% Negative Control

TABLE 19 Percentage of animals showing abnormal behavior followingchallenge (CBS 140425) Percentage of pigs Group # Pigs Vaccine showingabnormal behavior 1 13 Strain 10ΔfolT -  0% 10 logs - 2 dose 2 15 Strain10ΔfolT - 46.7%  10 logs - 1 dose 3 15 Placebo 100% Negative Control

TABLE 20 Percentage of animals expired or euthanized following challenge(mortality) (CBS 140425) Group # Pigs Vaccine Mortality (%) 1 13 Strain10ΔfolT -  0% 10 logs - 2 dose 2 15 Strain 10ΔfolT - 26.7%  10 logs - 1dose 3 15 Placebo - 100% Negative Control

TABLE 21 Percentage of animals with abnormal findings in brain uponnecropsy (CBS 140425) Percentage of pigs with Group # Pigs Vaccineabnormal findings in CNS (%) 1 13 Strain 10ΔfolT -   0% 10 logs - 2 dose2 15 Strain 10ΔfolT - 26.7% 10 logs - 1 dose 3 15 Placebo - 93.3%Negative Control

TABLE 22 Percentage of animals with abnormal findings in thoracic cavityupon necropsy (CBS 140425) Percentage of pig with lesions Group # PigsVaccine in thoracic cavity (%) 1 13 Strain 10ΔfolT - 23.1% 10 logs - 2dose 2 15 Strain 10ΔfolT - 33.3% 10 logs - 1 dose 3 15 Placebo - 93.3%Negative Control

TABLE 23 Percentage of animals from which S. suis was recovered frombrain swab (CBS 140425) S. suis recovered Group # Pigs Vaccine from CNSswab (%) 1 13 Strain 10ΔfolT -   0% 10 logs - 2 dose 2 15 Strain10ΔfolT -  6.7% 10 logs - 1 dose 3 15 Placebo - 73.3% Negative Control

What is claimed is:
 1. A recombinant ΔFolT mutant of a Streptococcussuis (S. suis) bacterium having reduced capacity to transport folatecompared to wild type, wherein said capacity has been reduced bydeletion or inactivation of a gene of the S. suis encoding folatetransporter (FolT) function.
 2. The recombinant ΔFolT mutant of claim 1having the capacity to synthesize folate.
 3. The recombinant ΔFolTmutant of claim 1 having reduced expression of FolT.
 4. The recombinantΔFolT mutant of claim 1 having a mutation or deletion of or in thepeptide domain FYRKP or an insertion in the peptide domain FYRKP.
 5. Therecombinant ΔFolT mutant of claim 1 deposited as “CBS 140425Streptococcus suis ΔFolT mutant” at the Centraalbureau voorSchimmelcultures at Aug. 19,
 2015. 6. The ΔFolT mutant of claim 1deposited as “CBS 143192 Streptococcus suis ΔFolT2 mutant” at theWesterdijk Fungal Biodiversity Institute at Aug. 25,
 2017. 7. Acomposition comprising the bacterium of claim
 1. 8. An immunogeniccomposition comprising the bacterium of claim
 1. 9. A vaccine comprisingthe bacterium of claim
 1. 10. A kit for vaccinating a pig, against adisease associated with a Streptococcus suis infection comprising: adispenser for administering a vaccine to the pig; the recombinant ΔFolTmutant strain according to of claim 1; and optionally an instructionleaflet.
 11. The ΔFolT mutant of claim 1, wherein amino acid R in apeptide domain FYRKP has been mutated.